Switching Cell Types Using CRISPR

Charles Gersbach, the Rooney Family Associate Professor of Biomedical Engineering and director of the Center for Biomolecular and Tissue Engineering at Duke University. Image courtesy of Duke University. Image Source: http://bit.ly/2cvRior

With CRISPr being the most trending tool in molecular biology today, researchers have now utilized the technique to convert connective tissue directly into neuronal cells.

Earlier in 2006, Professor Shinya Yamanaka from the Institute for Frontier Medical Sciences at Kyoto University discovered how to revert connective tissue cells into adult mature stem cells, from which they can be differentiated into any cell type. This breakthrough discovery won him the Nobel Prize for medicine six years later.

Since then, researchers have tried to convert cells between different cell types by introducing extra copies of “master switch” genes, that when introduced into the cells -produce proteins that are responsible for turning on genetic pathways for producing a particular cell type.

Now, researchers at Duke University have found a way to circumvent the introduction of these extra “master switch” genes by using CRISPr. A modification of the CRISPr technique is directly used to turn on the natural copies of the desired gene that is present in the genome.

The early results of the study published in the journal Cell Stem Cell demonstrates that the converted neuronal cells show a more persistent conversion as compared to the techniques that introduce the extra genes.

“This technique has many applications for science and medicine. For example, we might have a general idea of how most people’s neurons will respond to a drug, but we don’t know how your particular neurons with your particular genetics will respond,” said Charles Gersbach, the Rooney Family Associate Professor of Biomedical Engineering and director for the Center of Biomolecular and Tissue Engineering at Duke.

“Taking biopsies of your brain to test your neurons is not an option. But if we could take a skin cell from your arm, turn it into a neuron, and then treat it with various drug combinations, we could determine an optimal personalized therapy.”

“The challenge is efficiently generating neurons that are stable and have a genetic programming that looks like your real neurons,” says Joshua Black, the graduate student in Gersbach’s lab who led the work. “That has been a major obstacle in this area.”

This discovery comes at a time where traditional methods have accomplished this by using viruses as gene delivery vehicles to insert extra copies of the gene into the cell, in order to provide the “switch” to differentiate to other cell types.

“Rather than using a virus to permanently introduce new copies of existing genes, it would be desirable to provide a temporary signal that changes the cell type in a stable way,” said Black. “However, doing so in an efficient manner might require making very specific changes to the genetic program of the cell.”

In their study, Black, Gersbach, and colleagues have used CRISPr to intrinsically activate genes within the cells that naturally produce the master transcription factors that control the neuronal gene network.

In this case the team has tweaked the original CRISPr, which is a modified version of a bacterial defense system that targets and slices apart recognized sequences of the DNA of familiar invading viruses. Instead of the slicing, a gene activator is hitched onto the machinery that is involved in the gene identification, thereby activating the necessary genes.

The results from the laboratory tests conducted with mice show that, once activated by CRISPR, the three neuronal master transcription factor genes robustly activated neuronal genes, causing the fibroblasts to conduct electrical signals — a hallmark of neuronal cells. The cells also retained their neuronal activities after the CRISPr system was removed.

“When blasting cells with master transcription factors made by viruses, it’s possible to make cells that behave like neurons,” said Gersbach. “But if they truly have become autonomously functioning neurons, then they shouldn’t require the continuous presence of that external stimulus.”

“The method that introduces extra genetic copies with the virus produces a lot of the transcription factors, but very little is being made from the native copies of these genes,” explained Black. “In contrast, the CRISPr approach isn’t making as many transcription factors overall, but they’re all being produced from the normal chromosomal position, which is a powerful difference since they are stably activated. We’re flipping the epigenetic switch to convert cell types rather than driving them to do so synthetically.”

The obvious steps from here are to extend this system to human cells and increase the efficiency of the process.

“In the future, you can imagine making neurons and implanting them in the brain to treat Parkinson’s disease or other neurodegenerative conditions,” said Gersbach. “But even if we don’t get that far, you can do a lot with these in the lab to help develop better therapies.”

Arundithi holds a bachelor's degree in Biotechnology from SASTRA University, Tanjore, India. She is currently a PhD student in Nanyang Technological University. She works on the synthesis of graphene quantum dots for biomedical applications.